This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Niemann-Pick disease type C is a fatal hereditary lysosomal storage disorder resulting in progressive neurodegeneration and death, typically in the first decade of life. The NPC2 protein, deficient in Niemann-Pick type C2 disease, is a 132-amino acid protein that was previously characterized as a major secretory protein present in mammalian epididymis and shown to function as a cholesterol transfer protein. We have solved the 1.7 angstrom resolution crystal structure of bovine NPC2. NPC2 has an immunoglobulin-like fold stabilized by three disulfide bonds. The structure reveals a loosely packed region penetrating from the surface into the hydrophobic core that forms adjacent small cavities with a total volume of ~160 cubic angstroms. We propose that this represents the incipient cholesterol-binding site that dilates to accommodate an ~740 cubic angstrom cholesterol molecule. The structure provides an important foundation for functional analysis of NPC2 and provides a scaffold for interpreting mutagenesis data. The current structure is a starting point, not an end point. It has raised many important questions. Fro example, what is the binding site for cholesterol? What conformational changes are required for sterol binding? What is the mechanism of this conformational change? Are other protein partners involved in facilitating this transition? And, can the binding pocket accommodate sterols other than cholesterol? We propose to take a structural approach to investigating these issues. We propose to use structural information as a basis for analyzing sterol-binding by NPC2. Specifically, we aim to ) determine a high resolution x-ray crystal structure of NPC2 bound to cholesterol or a closely related analog, and 2) characterize the ligand binding pocket of NPC2 using a combination of computational and experimental approaches.